System of detection of pathogens 2

Question 1

Identify key methodologies that allow for the detection of pathogens by targeting specific genes

Answer 1

Nucleic Acid Amplification Tests (NAAT) are used to detect specific sequences of DNA or RNA, which can be helpful in identifying the presence of a particular pathogen in a sample

1) PCR:

• This technique amplifies specific gene sequences, allowing for the detection of even a small amount of a pathogen’s genetic material
• Denaturation: double-stranded DNA is heated typically to 95°C to break the hydrogen bonds between the bases, resulting in two separate DNA strands
• Annealing: When the temperature is lowered (typically to 50-65°C), the primers (short DNA fragments) bind to the complementary sequences on the DNA strands
• Extension/elongation: The temperature is increased to 72°C, the optimal temperature for Taq DNA polymerase. This enzyme adds dNTPs (deoxynucleoside triphosphates) to the 3’ end of the primer, based on the sequence of the template strand
• In the context of detecting pathogens, PCR is often used to amplify sequences unique to a specific pathogen, enabling identification even when only a small amount of the pathogen’s DNA is present in a sample
• Complex PCRs simultaneous amplification of multiple targets in a single reaction by using multiple sets of primers; useful when testing for multiple pathogens that might be causing similar symptoms

2) Quantitative PCR (qPCR):

• This variation of PCR measures the amplification of the targeted DNA segment in real-time (as the reaction is proceeding)
• allowing quantification of the starting amount of the DNA of interest
• A fluorescent probe is included in the reaction, which emits fluorescence when incorporated into the newly synthesised DNA - the increase in fluorescence correlates with the increase in DNA product
• The real-time monitoring of fluorescence throughout the PCR cycles allows the determination of the cycle at which the reaction moves into the exponential phase, which can be used to calculate the initial quantity of the target DNA (or cDNA in the case of RT-qPCR for RNA detection).
• For pathogen detection, this can provide information about the amount of pathogen present (the ‘load’), which can be useful for assessing severity of an infection or monitoring response to treatment

3) Reverse Transcription PCR (RT-OCR)

• Similar to PCR, but used to detect RNA (RNA viruses)
• involves an additional initial step where the RNA is reverse transcribed into complementary DNA (cDNA) by an enzyme called reverse transcriptase

4) Strand Displacement Amplification (SDA):

• SDA is an isothermal method, which means it operates at a constant temperature without the need for a thermal cycler
• The process starts with a DNA duplex that has a recognition site for a restriction endonuclease
• The endonuclease makes a nick in one strand at its recognition site, and a primer that is complementary to the region next to the nicked site is extended by DNA polymerase, displacing the downstream strand
• A second primer binds to the displaced strand, and DNA synthesis takes place from this primer, leading to the formation of a duplex DNA molecule
• The process repeats, leading to the amplification of the target sequence
• careful design of the primers and probes is essential to avoid non-specific amplification and false positives

5) Whole Genome Sequencing (WGS)

• involves determining the complete DNA sequence of an organism’s genome
• In the case of bacteria, this can help identify the specific strain and any drug-resistance genes it may carry

6) Next-Generation Sequencing (NGS):

• sequence millions to billions of DNA molecules simultaneously
• incredibly efficient, reducing the time and cost of sequencing compared to traditional methods (like Sanger sequencing)
• can be used to sequence complete genomes (as in WGS), or can be targeted to sequence-specific areas of interest

7) In Situ Hybridisation:

• uses a labeled complementary DNA or RNA strand (probe) to localise a specific DNA or RNA sequence in a portion or section of tissue
• The probe sequence is designed to be complementary to a sequence unique to the pathogen of interest, so if the pathogen is present in the sample, the probe will bind (or ‘hybridise’) to it

8) Microarrays:

• Microarrays (also called ‘DNA chips’) consist of a solid surface to which a large number of different single-stranded DNA fragments (probes) are attached at fixed locations
• The probes are designed to be complementary to sequences unique to different pathogens
• A pathogen’s DNA or RNA in the sample will bind to the corresponding probe on the array.

Question 2

Describe what makes a good gene target for pathogen detection

Answer 2

1) Specificity:

• The chosen gene must be unique to the pathogen being detected
• to avoid false-positive results
• The gene’s sequence should be well-conserved among different strains of the same pathogen but distinct enough from other pathogens

2) Stability:

• The gene target should be stable and not prone to frequent mutations, deletions, or insertions
• High genetic variability could lead to false negatives if the chosen primers or probes no longer match the gene’s sequence

3) Abundance:

• Ideally, the gene should be present in multiple copies in the genome of the pathogen to increase the sensitivity of the detection method
• particularly important in PCR-based methods, as the initial amount of target DNA can significantly affect the detection limit

4) Role in Pathogenicity:

• Genes that play a role in the pathogenicity of the organism can serve as good targets as their presence may indicate the potential for disease
• These may include virulence factor genes, toxin genes, or antimicrobial resistance genes

5) Availability of Sequence Data:

• The more sequence information available for the gene, the better, as this aids in designing specific and sensitive tests

Question 3

Explain how methods like MALDI-TOF produce pathogen-specific profiles at the protein level

Answer 3

Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS) is a powerful analytical technique used in the identification of microorganisms like bacteria and fungi

It is based on the analysis of proteins, mainly ribosomal, and their mass-to-charge ratio (m/z), which provides a unique spectral signature or “fingerprint” for each organism

1) Sample Preparation:

• A tiny amount of the microorganism (colony or culture) is smeared onto a metal plate
• This biological sample is then overlaid with a matrix solution, a small organic molecule that helps with the ionisation process
• The matrix absorbs energy from the laser, and it helps in desorbing and ionising proteins from the microorganism

2) Laser Desorption and Ionisation:

• Once the sample is prepared and placed in the instrument, a laser beam is focused onto the sample spot, causing desorption and ionisation of the sample proteins
• These ionised proteins are now ready to be accelerated in the electric field

3) Time of Flight and Detection:

• The ionised proteins are then accelerated in an electric field according to their charge, and they fly down a long tube (the flight tube) to the detector
• Lighter ions or those with a higher charge reach the detector faster. The time it takes for ions to reach the detector is measured, hence “Time-Of-Flight”

The MALDI-TOF MS output is a spectrum with signal intensity plotted against the mass-to-charge ratio (m/z) of the detected ions

The specific pattern of peaks forms a unique spectral signature that can be used to identify the microorganism

The collected spectrum is then matched against a database of known microbial spectra to identify the species

Thus, MALDI-TOF MS allows rapid, reliable, and cost-effective identification of microorganisms at the species level and sometimes even at the strain level

Question 4

Define biomarker of virulence

Answer 4

A “virulence biomarker” is a specific attribute of a pathogenic organism that not only contributes to its capacity to induce disease but can also be quantified and monitored

1) Definition:

• A virulence biomarker refers to a specific feature, typically a molecule or structure, associated with a pathogen that contributes to its virulence - its ability to cause disease
• 2) Examples:
• Examples of virulence biomarkers may include toxins produced by the pathogen, enzymes aiding in host cell invasion, surface proteins facilitating adhesion to host tissues, or elements contributing to antimicrobial resistance
• 3) Clinical Relevance:
• Clinically, virulence biomarkers can offer critical insights into the severity or progression of an infection
• For instance, detection of a certain toxin or the gene encoding it, typically associated with severe disease forms, could signal a high-risk infection requiring intensive treatment

4) Dependent Factors:

• The efficacy of a virulence biomarker can depend on several variables such as the specific pathogen involved, host species, the nature and site of the infection, and the available methods for biomarker detection and quantification

Question 5

Identify the fundamental elements behind key methodologies that allow for the detection and characterisation of pathogens at the whole genome level

Answer 5

Next-Generation Sequencing (NGS), also known as high-throughput sequencing, is a powerful method for the detection and characterisation of pathogens at the whole-genome level

1) Sample Preparation:

• extraction of nucleic acids from the sample
• For RNA viruses, reverse transcription is necessary to convert RNA into complementary DNA (cDNA)

2) Library Preparation:

• Following nucleic acid extraction, the next step involves preparing a sequencing library
• This includes fragmentation of the DNA or cDNA into smaller pieces, then attaching adapters to both ends of these fragments
• The adapter sequences are required for the DNA fragments to bind to the flow cell where sequencing takes place, and they also allow for PCR amplification of the library
• In some protocols, unique ‘barcode’ sequences might also be added to each sample, which allows for multiple samples to be pooled and sequenced together in the same run

3) Sequencing:

• The prepared library is then sequenced on an NGS platform
• generate a massive number of sequencing reads in parallel
• Illumina (sequencing by synthesis), Ion Torrent (semiconductor sequencing), PacBio (single-molecule real-time sequencing), and Oxford Nanopore (nanopore sequencing)

4) Bioinformatics Analysis:

• The raw sequence data generated through NGS is large and complex, requiring specialized bioinformatics tools for analysis
• Quality control checks are performed to ensure that the data is of good quality
• The sequence reads are then aligned to reference genomes (if available) or assembled de novo
• The alignment allows for the identification of genetic variants like single nucleotide polymorphisms (SNPs), insertions, and deletions
• This information can be used to characterise the pathogen at the strain level, identify virulence factors, and predict antibiotic resistance

5) Interpretation:

• The findings from the NGS data analysis can provide in-depth insights into the genetic makeup of the pathogen, aiding in diagnostic decisions, infection control measures, and epidemiological studies
• For example, it can inform about the source of an outbreak, track the spread of the pathogen, or detect the emergence of new strains

Question 6

Describe the requirements, advantages and disadvantages of sequencing methods

Answer 6

1) Sanger Sequencing

Requirement:

• requires purified DNA of the region of interest
• Once purified, the DNA is used to create a sequencing library which is then loaded onto a gel for electrophoresis
• also requires fluorescently labelled dideoxynucleotides, which terminate the DNA sequence, allowing for the determination of the DNA sequence based on the colour emitted upon laser excitation

Advantages:

• one of the most accurate sequencing methods available (error rate of less than 1 in 10,000 nucleotides)
• is a straightforward method and does not require complex computational analysis, making it accessible to most labs

Disadvantages:

• time-consuming, especially when sequencing large genomes, as it is a low-throughput method
• the cost per base of sequencing is high compared to next-generation methods

2) Next-Generation Sequencing (NGS):

Requirement:

• NGS methods require purified DNA or RNA, which is then fragmented into smaller pieces
• These fragments are then used to create a sequencing library, which is loaded onto a flow cell where the sequencing occurs
• Bioinformatics tools and significant computational resources are needed to assemble and interpret the enormous amount of data generated

Advantages:

• high-throughput capability, which allows for the simultaneous sequencing of millions to billions of DNA fragments
• An ideal choice for whole-genome sequencing, transcriptomics studies, and metagenomics studies

Disadvantages:

• requires considerable computational resources for analysis and storage
• cost per base is lower, the overall cost can still be high, especially when factoring in data analysis and storage costs